Inhibition of Na⁺-K⁺-ATPase activates Na⁺-K⁺-2Cl^- cotransporter activity in cultured ciliary epithelium

⁸⁶Rb uptake experiments were conducted to measure Na⁺-K⁺-ATPase activity and Na⁺-K⁺-2Cl^- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na⁺-K⁺- 2Cl^- cotransporter was supported by the observation of a bumetanide- sensitive ⁸⁶Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na⁺-K⁺-2Cl^- cotransporter and Na⁺-K⁺-ATPase accounted for ~46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistent uptake accounted for 9%. Inhibition of the Na⁺-K⁺- ATPase had a stimulatory effect on Na⁺-K⁺-2Cl^- cotransporter activity, which was dependent on the extent and duration of Na⁺-K⁺-ATPase inhibition. Ouabain treatment stimulated the potassium (⁸⁶Rb) efflux rate and reduced intracellular potassium ([K](i)). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na⁺-K⁺-2Cl^- cotransporter. We conclude that Na^--K⁺- ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na⁺-K⁺-2Cl^- cotransporter stimulation caused by the fall of [K](i) overrides the tendency of increased cellular sodium to inhibit the cotransporter.